How to keep stickleback

We use standalone 100 L aquaria to keep and raise stickleback. The fish grow well under these conditions provided densities are kept low, and the risk of death from chlorine spikes in the municipal water source is minimized. The greatest dangers are (1) kick-starting the nitrogen cycle when setting up a new tank, and (2) preventing transfer of disease from tank to tank, particularly from wild fish to lab-raised stocks.

Set up a new aquarium

Set up the aquarium in a cool room. 17 degrees C is perfect for our local populations. Above 20 is dangerous.

Dechlorinated tap water is fine. Add extra conditioning, such as Prime or Amquel, as backup dechlorination in case of a spike in chlorine in the water supply. Do not use water obtained from copper pipes, the copper ions will kill the fish. Add baking soda if pH is below 7 (Vancouver water is sometimes acidic).

To aid fish health, add 100-200 g of synthetic sea salt (e.g., Deep Ocean Synthetic Sea Salts) to each each 100 L aquarium. Add even more, 500 g, if you are raising crosses between freshwater and Little Campbell River marines (LCM), which tend to develop poorly at low salinity. This will bring salt concentration up to about 5 ppt.

Add a kg or so of coarse washed limestone to the tank. This keeps the water hard, which is best for minimizing disease. Many of our populations live in hard water lakes. Ordinary board chalk or powdered dolomite will substitute if you are in a rush.

One airstone per 100 L aquarium is fine (you don't need to add one if you are also using a sponge filter). Keep the flow light when the fish have just hatched, because they are not strong swimmers.

We use sponge filters for babies and both sponge filters and power filters for juveniles and adults. The foam inserts of the power filters last a long time but need periodic cleaning. Buy bulk activated charcoal and roll some up in doorscreen with elastics to fit the space in the filter. We put a bit of angel hair in the filter box for extra surface area and filtration of fine particulates.

Be careful using power filters with babies, as the flow can be too great. Put a sponge or fine screen mesh on the end of the intake pipe to prevent sucking up fish. Clean filter inserts or replace charcoal insert (but not both) every two weeks. Squeeze the sponge filters every two weeks or so to keep the sponges from plugging up.

Kick-start the nitrogen cycle

This is the greatest challenge when starting up a tank for fish. An inadequate nitrogen cycle is the greatest killer of lab fish, and the ruination of many experiments. Fish often die on weekends when there are few eyes watching. The death of the first one or two fish only makes things worse if they are not removed immediately, and can precipitate a mass extintion.

The problem is that fish waste is toxic. Ammonia starts rising almost as soon as you introduce the fish to the tank.  Soon after, nitrite starts building as bacteria oxidize the ammonia. Nitrite is also toxic to the fish. Eventually, bacteria will break down the nitrite to nitrate, which is relatively harmless and can be kept at low levels by regularly changing some of the water in the tank.

The main purpose of the filters is to provide a surface for the bacteria and flow-through to put them in contact with all the water in the aquarium. However, it takes at least a month for the bacteria to reach sufficient numbers to handle all the waste produced by the fish. Even established tanks may experience fluctuations in bacteria numbers, resulting in accumulation of toxic waste products.

Here are some strategies to avoid fish death by toxic waste:

  • Set up new tanks at least one month before introducing your valuable fish. While you wait, keep a goldfish, sculpin, or a couple of non-essential stickleback in the tank to help get the nitrogen cycle going before you replace them with your valuable fish. (Another technique is to add ammonia daily directly to a fishless tank and keep testing the water until nitrites drop to zero).
  • If you don't have the luxury of a one-month advance, but must throw your fish straight into a new, sterile tank, then you will need to change the water frequently to keep waste levels down while the nitrogen cycle gets started. Try replacing 1/3 to 1/2 of the water every two days for the first two weeks at least. Add Amquel or other conditioner to help reduce the waste (note: choose a product that removes nitrite as well as ammonia -- not all of them do so).
  • Make arrangements for water changes on weekends if you are not around to do it yourself, especially long weekends.
  • When you add fish to a new tank, simultaneously add an old sponge from a working aquarium to introduce the bacteria. (Warning: This can also transmit disease between tanks.) Or, use a filter from the fish-free nitrogen cycle tank, if one is available. This will give your nitrogen cycle a head start, though you will still need to monitor its development. 
  • Keep fish densities low, even after the nitrogen cycle has started to work. A 100 L tank can handle about 15 adult fish when everything is going well (maybe even 20 if you are not dealing with large benthic or marine fish). If you go higher, be prepared for the higher risk and more water changes. Go lower if the fish are irreplaceable.
  • Test ammonia and nitrites frequently. But don't wait for the test results to act, or place too much reliance in them, especially if you have set up a new tank. Waste can accumulate rapidly. 
  • Even after the nitrogen cycle is working, change about half the water in every tank each week.
  • Remove dead fish immediately. Take special care on weekends, if you are alone in the lab. You might be able to avert disaster this way.
  • Do not overfeed fish. If the fish food that drops to the bottom of the tank isn't eaten within a few minutes, siphon the excess away.
  • If you are raising fish from eggs, you might end up with as many as 200 juveniles from one clutch in a single aquarium. This is unsustainable. In this case, families should be divided among muliple tanks as soon as the young fish are large enough to tolerate a move.
  • Make sure your filters are working properly. Restart them immediately if there has been a power outage.
  • Once the nitrogen cycle is going swimmingly, clean part of the contents of your filters regularly (i.e., the charcoal or the sponge, but not both). Don't clean the entire filter, as this may knock back the bacteria numbers too greatly.

Make crosses

Obtaining eggs from females:

A female ready to spawn can be identified by her abdomen shape at the cloaca. If ready to lay eggs, one can almost see the first egg; at this point her abdomen at the cloaca is sharply angled, almost like the corner of a box. The eggs should come after gently squeezing her body above and forward of the egg mass and, while maintaining pressure, sliding your fingers posteriorly. The eggs, when they appear, will stick to one another in a clump. If they disaggregate then the female was not ready and you should throw the eggs out. Keep the eggs covered with water, but keep them near the surface for oxygen.

Testes:

We haven't developed a method to extract sperm without killing the male. Extract testes from the male after giving him an overdose of anaesthetic. If you do not actually require separate crosses, you can fertilize several clutches with the testes from one male. Male sperm will keep in Hanks solution for a few days; see Storing sperm. Shred the testes with tweezers and stir around the egg mass. Remove the testes after a few minutes otherwise they might decay and attract fungus. It is worthwhile to check that the eggs are fertilized and begin development (the easiest way to tell is under a low-power microscope---look for the separation of outer and inner membranes).

Store sperm for up to 6 weeks

Materials needed

  • 49.5mL of Ginsburg's ringer solution (a buffer solution - recipe is below)
  • 0.25mL of Gibco antibiotic/antimycotic (Invitrogen cat# 15240-096, 100x
    concentration),
  • 0.25mL of Gentamycin sulfate hydrate, (Invitrogen cat# 15750-060, 10ml, 50mg/ml
    liquid)
  • 1 falcon tube.

Preparation of the storage solution

  1. measure out 49.5mL of Ginsburg's ringer solution and pour into a falcon tube.
  2. pipette 0.25mL of both the Gibco and Gentamycin solutions into the Ginsburg's solution in the falcon tube.
  3. close cap and shake well in order to thoroughly mix solution

Sperm storage

  1. after removing the testes from the male stickleback place each testis (or portion of testis) in it's own Eppendorf tube.
  2. pipette in enough Ginsburg's solution to fill half the tube.
  3. make sure that each testis (or portion of testis) is completely submerged in the solution (it should sink to the bottom).
  4. store in fridge
  5. replace with fresh Ginsburg's solution every 7-10 days for storage up to 6 weeks.

Recipe for Ginsburg's ringer solution

  1. To 900 mL of ddH2O, Add:
    6.6 g NaCl
    0.25g KCl
    0.3 g CaCl2
  2. Add 0.2g NaHCO3 last
  3. Mix well, and bring up to 1 L
  4. Autoclave


Raise crosses

Raise eggs

We use either of two approaches to raise the eggs to hatching. The egg-tank method involves raising eggs for the first 7 days in small 5 gallon fish tanks and then transferring the eggs to standard 100 L tanks just before they hatch. The 7 day period assumes that the rooms are at 17 degrees. If the room is warmer they may hatch sooner. Egg-tanks should be cleaned out and made fresh every 2 weeks. The big-tank method involves raising eggs entirely in the 100 L tanks.

In either case, place the eggs in a yoghurt cup having mesh screen on the bottom. Suspend the cup from the side of the aquarium so that the eggs are well submerged. Door screen is best, because the fry drop through after hatching (another reason to make sure to transfer eggs from egg-tanks to 100 L tanks before hatching begins). Put an airstone nearby to provide oxygen and maintain a current around the eggs. Avoid fine streams of bubbles directly underneath the eggs because air may get trapped under in the egg mass, bringing it to the surface, where the eggs may dry out.

The water in the tank should be dechlorinated. Adjust the pH to 7 (using baking soda or pH adjuster). Add 100-200 g of synthetic sea salt (e.g., Deep Ocean Synthetic Sea Salts) per 100 L water. Add at least 500 g/100 L if you are raising crosses between freshwater and Little Campbell River marines (LCM), which tend to develop poorly at low salinity. Also add Methylene Blue solution to the tanks to help reduce fungus attack. Mix the powder thoroughly with water in a falcon tube and then add a few drops to the tank so that the water color is a pale blue (don't add the powder directly to the tank). The 100 L tanks should be set up at least a day in advance of the egg transfer, so the filters have time to clean the water and help dissolve/mix the salt and baking soda. Use only a sponge filter in the tank. Make sure power filters are shut off. Use only sponge filters until young are large enough to handle the current and suction of a power filter.

Tend the eggs daily and remove clots of fungus that appear. Be careful not to tear nearby eggs. You will never manage to be free of fungus, but plenty of aeration and the methylene blue will help to avoid the worst. Our experience is that if fungus is out of control in a clutch of eggs only a couple of days after fertilization, then the clutch probably wasn't fertilized after all.

Raise young fry

When the young hatch they will sink to the bottom and stay there for a couple of days. Then they will swim up and gulp some air from the surface to establish neutral bouyancy. Make sure that the sponge filters and airstones are not bubbling too vigorously at this time (keep the power filters off until the babies are older). After a few days you will start to see the tiny babies hanging out together off the bottom, especially in the corners of the aquarium.

Start adding a squirt of infusoria to tanks the day they hatch. You may also add a few drops of pet-shop liquid food for egg-layers. They don't eat the stuff; rather, they eat the paramecium and possibly some bacteria. Continue adding infusoria for about 5 days after hatch, by which time all the fry will be free-swimming.

About 2 days after hatching, start feeding them microworms, if available, and small quantities of first-instar brine-shrimp nauplii. Add enough brine shrimp so that after they have fed their bellies are orange and swelled. It is best to feed twice daily until they are a few weeks old, but once daily will also work if you don't mind the slower growth. When the young reach about 2 cm, start feeding them frozen bloodworms. We continue to feed them a small amount of brine shrimp nauplii as well, even as adults.


Troubleshooting

Nitrites are high

Usually a problem in recently cleaned tanks, or overstocked tanks. Monitor N levels monthly and note level on tank. When a tank has detectable nitrites:
  • change 1/2 of the water immeidately
  • add 5-15 ml of Prime (more directions on bottle of Prime)
  • add another filter
  • move some fish to another tank
  • recheck N next day, continue above steps until problem solved
Continue to replace 1/3 of the water in the tank daily until nitrites are undetectable.

Filters not working/water not flowing properly

Check whether it's a single filter or a bunch in one area. If single:
  • check if it's plugged in (make sure it's not a new baby tank and therefore supposed to be unplugged)
  • check power bar is working
  • jiggle motor fan with finger to get it working again (sometimes they jam)
  • check u-tube and downspout are attached properly near the motor and are sucking up water (sometimes the filter is on, but no water is cycling – especially important to check after cleaning filters)
  • check that the downspout isn't blocked (plant material/dead fish/mesh clogged with dirt)
  • check for broken parts and discard broken bits, re-assemble filter with new part (there's boxes of filter parts in one of the rooms, including motors, bodies, fans etc)
  • follow the power to where it doesn't work (the power bar has probably gotten wet or there's a short somewhere)
  • Jump start all filters that don't automatically restart themselves, but do it after you've sorted the problem out or you'll be restarting things all day.


Rooms Are Warm

Horrifically bad if this happens and needs to be fixed immediately or all the fish will die (Not a good thing). Needs to be fixed within the day.
  • Check that the air conditioner is on and working.  If it's broken or blowing out warm air, check the settings. If they're set above 20, turn down.  If they're set below 20, something's wrong with the air conditioner. Try turning it off and then back on.  Wait to see if it starts blowing cool air. If it's during the week, hop down to see Don or Bruce in the shop and ask them to have a look. If they can't fix or they're not around, phone plant ops ‘trouble calls' (ext 2-2173) immediately, and tell them it's crucial (for some reason problems seem to happens on a weekend or late Friday afternoon).
  • If air-conditioner is working (blowing out cold air), make sure all the doors and windows to the room are closed. On hot days the rooms will heat up if you leave the doors open.
  • check the insulation on the windows and fix if it comes apart (duct tape, Styrofoam, cardboard and garbage bags are the current insulation)

All or most fish in one tank are suddenly dead:

  • Not a good sign.  Move any remaining fish to a new tank while you sort out what happened.
  • Look at the history of the fish and tank.
  • New Tank:
    • Potential N problem, check and fix
    • If the tank is full of dead babies it could be genetic or food related, but check nitrite, pH and ammonia levels (buy some indicator strips, we don't have any right now). Don't put any more babies in here until you're absolutely sure none have survived (either empty and refill tank, or add some hungry lab-raised, large adults to eat remaining bb's), and you're sure it wasn't tank related. 
  • Established Tank:
    • Check dead fish for signs of ick or other disease, if tank has ick, do not move remaining fish.  Take out charcoal from filter and throw in garbage, then add 27 drops of Ick treatment (blue jug under main rm sink), follow directions on back of jug for follow up treatment
    • If tank doesn't have ick, Move any remaining fish to a new tank.  Might be NH4 spike, N spike, or some other chemical that got into the tank.  I actually have never been able to figure out what went wrong when this happens (unless it turns out to be N) because it only happens once a year with lab-raised fish
    • If it's N, don't clean the tank, but do sort out the N problem until it settles down, then you can re-use for fish.  You can empty half the tank and re-fill with water, just don't clean completely (to avoid ‘new tank' N issues)
    • If it's not N, empty most of the tank and re-fill, anyway.  Change the charcoal.  Clean only if it's had wild fish in it.
  • Tank with Wild Fish
    • If the fish have been brought back recently (less than 2 days), it's probably something that happened to them when in transit (too cold, too crowded, too hot, not enough air, etc), or the tank and water they're used to are wildly differing pH's.  Try to match the pH of the tank to the pH of their lake (within 0.5 – indicator strips are accurate enough).  Also check the ammonia and nitrite levels in the tanks just to be sure, add 5-15 ml of prime as precaution.  Decide whether to move them or not.
    • If fish are wild, but have been in the lab for more than 2 days, follow the same directions listed for an established tank